EP2794918A1 - Diagnostic de la stéatohépatite - Google Patents

Diagnostic de la stéatohépatite

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Publication number
EP2794918A1
EP2794918A1 EP12813365.9A EP12813365A EP2794918A1 EP 2794918 A1 EP2794918 A1 EP 2794918A1 EP 12813365 A EP12813365 A EP 12813365A EP 2794918 A1 EP2794918 A1 EP 2794918A1
Authority
EP
European Patent Office
Prior art keywords
krt23
dna
methylation
sample
steatohepatitis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12813365.9A
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German (de)
English (en)
Inventor
Kurt Zatloukal
Karl KASHOFER
Michael Trauner
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Medizinische Universitaet Graz
Original Assignee
Medizinische Universitaet Graz
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Priority to EP12813365.9A priority Critical patent/EP2794918A1/fr
Publication of EP2794918A1 publication Critical patent/EP2794918A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4742—Keratin; Cytokeratin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the invention relates to biomarkers for distinguishing stea- tohepatitis from steatosis.
  • Fatty liver diseases comprise a spectrum of severity ranging from simple steatosis over steatohepatitis to cirrhosis and hepatocellular cancer (HCC) .
  • HCC hepatocellular cancer
  • T2DM type 2 diabetes mellitus
  • Up to 30% of the general population is affected by non-alcoholic fatty liver disease (NAFLD) , reach ⁇ ing up to 70% among diabetic patients.
  • NAFLD non-alcoholic fatty liver disease
  • steato ⁇ sis and steatohepatitis in obese patients undergoing bariatric surgery is as high as 76% and 37%, respectively.
  • Steatohepatitis develops in about 20% of alcoholics and up to 50% of T2DM who are also obese (BMI > 30) .
  • steatohepatitis While simple steatosis has a relatively benign course and is principally reversible, steatohepatitis carries a poor prognosis and can lead to severe liver damage with progression to cirrho ⁇ sis and HCC.
  • Conventional non-invasive markers such as serum transaminases correlate poorly with the risk of development as well as progression of liver disease, and currently available routine liver tests may even be unremarkable in a significant proportion of patients with steatohepatitis. Therefore, in cur ⁇ rent standard clinical practice, non-invasive serum and imaging markers do not allow the distinction of relatively benign fatty liver from progressive steatohepatitis. This situation results in underdiagnosis and undertreatment of these disorders.
  • a major unsolved problem is the marked difference in the individual risk to develop steatohepatitis and to progress to cirrhosis (e.g., only 20% of heavy drinkers or 50% of obese type II diabetic patients develop steatohepatitis; Hispanics and Cau ⁇ casians are more susceptible than Afro-Americans) .
  • cirrhosis e.g., only 20% of heavy drinkers or 50% of obese type II diabetic patients develop steatohepatitis; Hispanics and Cau ⁇ casians are more susceptible than Afro-Americans.
  • the factors responsible for disease progression across the spectrum of fatty liver disease are poorly understood. Why some patients are protected against developing steatohepatitis or simple stea ⁇ tosis, while others are not, is still unclear.
  • liver biopsy Diagnosis of steatohepatitis is difficult and currently only possible by liver biopsy (for example, NASH refers to findings on liver biopsy in patients with steatohepatitis in the absence of significant alcohol consumption) . Therefore, liver biopsy re ⁇ mains the only means of assessing the presence and extent of specific necroinflammatory changes and fibrosis in steatohepati ⁇ tis. However, firm recommendations of when to perform a liver biopsy in the routine clinical setting have not yet been devel ⁇ oped. The use of surrogate markers, such as aminotransferases and fibrosis markers, are not adequate for monitoring diseases as is necessary for clinical studies.
  • WO 2004/055520 Al discloses a method of diag ⁇ nosing NASH.
  • WO 2010/045470 A2 discloses determining the risk for developing a hepatic disorder by determining the level of expression of certain makers.
  • the present invention is specifi ⁇ cally aimed to provide means and methods for differentiation be ⁇ tween the clinically "benign" simple steatosis and steatohepati ⁇ tis that might progress to liver cirrhosis and liver cancer.
  • the present invention provides the use of methyl ⁇ ation of the keratin 23 (KRT23) DNA as a marker for distinguishing between steatosis and steatohepatitis.
  • the methylation of the KRT23 gene is a highly reliable molecular marker for dis ⁇ tinguishing steatosis from steatohepatitis.
  • methyla ⁇ tion of the KRT23 gene can also be used for monitoring the de ⁇ velopment from steatohepatitis to HCC .
  • KRT23 methylation can al ⁇ so be used together with other markers for steatohepatitis and/or HCC for further optimising diagnosis of such liver diseases .
  • the human KRT23 gene (2 alternative transcripts: NC_000017.10, NT_010783.15; Seq.ID.No.l) is located on chromo ⁇ some 17 (17q21.2; reverse strand) and spans from nucleotide 39, 078, 952 bp to 39, 099, 836 bp (chrl7: 39, 078, 952 - 39, 099, 836). It has therefore a size of 20,885 bases.
  • methylation status of the KRT23 DNA can be determined by determining presence or absence of methyla ⁇ tion at any CpG motif (a "CG" dinucleotide) in the gene se ⁇ quence.
  • methylation status of the KRT23 DNA is preferred, however, to determine presence/absence of methylation in a region containing more than one CpG motif, preferably in a region with at least 5, more preferred with at least 10, especially with at least 20 CpG motifs.
  • Specifically preferred regions for determining the methylation status of the KRT23 DNA according to the present invention are the exon re ⁇ gions.
  • the KRT23 gene contains 9 exons :
  • a preferred region where methylation is determined according to the present invention is the promoter region defined as the region 6000 bases upstream of the transcription start (from nucleotide 39,093,836 to nucleotide 39,099,836 (chrl7: 39,093,836- 39,099,836 (Seq. ID. No. 2).
  • the determination of the degree of KRT23 methylation is spe ⁇ cifically suitable for diagnosing NASH, since KRT23 methylation levels are significantly decreased in NASH patients compared to healthy individuals or individuals having steatosis.
  • a demethyl- ation of the KRT23 DNA (compared to a KRT23 DNA from a healthy person (i.e. a person not having steatohepatitis) ) is therefore indicative for steatohepatitis.
  • Demethylation is specifically present if 10 % or more, preferably 20% or more, especially 30 % or more of the methylated CpG motifs of the healthy KRT23 DNA region determined are demethylated in the DNA in the sample.
  • the method according to the present invention is advanta ⁇ geous compared to current diagnosis (grading, staging) of stea- tohepatitis which relies on liver biopsy as a diagnostic gold standard for differentiation between "simple" steatosis and (N)ASH.
  • Routine biochemical serum tests liver function tests
  • liver function tests currently available underestimate the occurrence and severity of steatohepatitis .
  • the degree of liver fibrosis may be a crude predictor for the development of liver cirrhosis, more so ⁇ phisticated individual risk markers/profiles for the development of cirrhosis and hepatocellular cancer are required.
  • a specific aspect of the present invention relates to a method for diagnosing steatohepatitis in a sample of a human body fluid or a human tissue sample comprising the determination of the methylation of the KRT23 DNA in this sample and diagnos ⁇ ing steatohepatitis, if the methylation of the KRT23 DNA is de ⁇ creased compared to a sample of this human body fluid or tissue sample from a person with no steatohepatitis.
  • the body fluid is blood or a blood derived sam ⁇ ple, preferably a serum or a plasma sample.
  • a blood derived sam ⁇ ple preferably a serum or a plasma sample.
  • Measuring KRT23 methylation in DNA in blood or a sample derived from a blood sample is easily adaptable to larger numbers of samples and may therefore be established for routine testing, preferably for de ⁇ termination of the methylation degree of the KRT23 gene in free circulating DNA in blood, especially in plasma or serum samples.
  • the present method can also routinely be applied for tissue samples, especially liver tissue samples (e.g. taken from liver surgery) or liver biopsy samples (e.g. taken by usual liver biopsy needles, historical samples or sam- pies from necropsies) .
  • liver tissue samples e.g. taken from liver surgery
  • liver biopsy samples e.g. taken by usual liver biopsy needles, historical samples or sam- pies from necropsies
  • Determination of the methylation of the KRT23 gene is easily possible for a person skilled in the art. Both, preparation of DNA in the sample for determination of methylation as well as establishing the amount/degree of methylation are routine meth ⁇ ods in the present field of technology. For example, there are several commercially available kits for extracting DNA from blood samples, as well as kits for determining the methylation of DNA (e.g. using the bisulfite treatment with subsequent PCR amplification) .
  • the present invention refers to the use of the (determina ⁇ tion of the) methylation (degree) of the KRT23 gene in a method for diagnosing steatohepatitis.
  • KRT23 is a gene/protein which is widely known andomme ⁇ gated; determination of the degree of methylation of the KRT23 DNA is therefore easily possible by using standard techniques well available to a person skilled in the art.
  • DNA methylation of the KRT23 gene can be detected and quan ⁇ tified by any method commonly used in the art, for example, methylation-specific PCR (MSP) , use of restriction enzymes with activity governed by methylation status, use of antibodies spe ⁇ cific for methylated nucleotide bases, polynucleotide sequenc ⁇ ing, bisulfite treatment and sequencing, pyrosequencing, and absolute quantitative analysis of methylated alleles (AQAMA) .
  • MSP is a technique whereby DNA is amplified by PCR dependent upon the methylation state of the DNA.
  • Determination of the methylation state of a nucleic acid includes amplifying the nucleic ac ⁇ id by means of oligonucleotide primers that distinguish between methylated and unmethylated nucleic acids.
  • MSP can rapidly as ⁇ sess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation- sensitive restriction enzymes.
  • This assay entails initial modi ⁇ fication of DNA by sodium bisulfite, converting all unmethylat ⁇ ed, but not methylated, cytosines to uracils, and subsequent am ⁇ plification with primers specific for methylated versus un ⁇ methylated DNA.
  • MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island lo ⁇ cus. MSP eliminates the false positive results inherent to pre ⁇ vious PCR-based approaches which relied on differential re ⁇ striction enzyme cleavage to distinguish methylated from un- methylated DNA. This method is very simple and can be used on small amounts of samples. MSP product can be detected by gel electrophoresis, CAE (capillary array electrophoresis) , or real ⁇ time quantitative PCR.
  • Bisulfite sequencing is widely used to detect 5-MeC (5- methylcytosine) in DNA, and provides a reliable way of detecting any methylated cytosine at single -molecule resolution in any sequence context.
  • the process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine under ⁇ goes conversion to uracil while 5-MeC remains unreactive.
  • the level of DNA methylation may be represented by a methyl ⁇ ation index as a ration of the methylated DNA copy number to the sum of the methylated DNA copy number and the unmethylated DNA copy number, a ratio of the methylated DNA copy number to the unmethylated DNA copy number, or the like.
  • a "normal sample” is a sample prepared from a normal subject, es ⁇ pecially a normal body fluid, normal blood or blood derived sam ⁇ ple, such as a serum or plasma sample.
  • the KRT23 DNA is free circulating DNA in blood, especially in plasma or serum samples.
  • Preferred methylation determination methods are methods employing the bisulfite treatment.
  • the methylation degree of the KRT23 DNA in the sam ⁇ ple is compared with a reference value for this amount with a known status concerning steatohepatitis and/or steatosis, i.e. a value which is known to be a healthy value (i.e. a value not af ⁇ fected by steatohepatitis) or which is known to be a diseased status with respect to steatohepatitis/steatosis, preferably of a given/defined stage of steatohepatitis.
  • a known status concerning steatohepatitis and/or steatosis i.e. a value which is known to be a healthy value (i.e. a value not af ⁇ fected by steatohepatitis) or which is known to be a diseased status with respect to steatohepatitis/steatosis, preferably of a given/defined stage of
  • standard or control samples are all useable in principle for comparison with the sample of unknown status concerning steatohepatitis and/or steatosis diagnosed according to the present invention.
  • standard or control samples can be taken e.g. from a human subject with negative di ⁇ agnosis concerning steatohepatitis and/or steatosis or undetect ⁇ able ongoing steatohepatitis development.
  • control sam- pie, standard sample or reference sample is said to be compara ⁇ ble to the sample that is taken from a human subject being sus ⁇ pected to be afflicted by ongoing steatohepatitis development according to the present invention, this means that both samples have been derived and treated equally.
  • the sample in both cases may e.g. be a blood derived sample which has been further treated in a way to allow for determination of methylation of the diagnostic marker gene as mentioned above.
  • Blood plasma is the yellow liquid component of blood, in which the blood cells in whole blood would normally be suspend ⁇ ed. It makes up about 55% of the total blood volume. It is most ⁇ ly water (90% by volume) and contains dissolved proteins, glu ⁇ cose, clotting factors, mineral ions, hormones and carbon diox ⁇ ide. Blood plasma is prepared by spinning a tube of fresh blood in a centrifuge until the blood cells fall to the bottom of the tube. The blood plasma is then poured or drawn off. Blood plasma has a density of approximately 1.025 kg/1. Blood serum is blood plasma without fibrinogen or the other clotting factors (i.e., whole blood minus both the cells and the clotting factors) .
  • the diagnostic methods according to the present invention may also be carried out in saliva, bladder washing, semen or urine samples, especially urine samples.
  • the diagnostic fine tuning for established clinical practice will work with sequence listings for defining the methylation differences in the part of the KRT23 gene for which methylation status has been determined according to the present invention
  • the samples are preferably blood, plasma, serum, urine, semen or saliva samples: also here suitable comparison values are derived from samples blood, plasma, serum, urine, se ⁇ men or saliva, respectively, from a patient with has a healthy status, preferable samples taken (earlier) from the same pa ⁇ tient. On the other hand, the comparison may also be taken to establish known methylation patterns of KRT23 DNA in the sample taken. As usual in human medical diagnosis, absolute limiting value can be defined for each of the samples to determine dif ⁇ ference between healthy (steatosis) and steatohepatitis and be ⁇ tween different stages steatohepatitis or even HCC .
  • the level of methylation of KRT23 DNA assessed according to the present invention is measured and is compared with the level of expression of methylation of KRT23 DNA from other samples.
  • the comparison may be effected in an actual experiment; or in ⁇ tellectually (by comparison with known reference values) ; or virtually (e.g. automatically in silico) .
  • the methylation level also referred to as methylation pattern or methylation signature (methylation profile)
  • a meaningful (i.e. statistically significant) difference in the level of methylation is measurably different, there is according to the invention a meaningful (i.e. statistically significant) difference in the level of methylation.
  • the difference in methylation of KRT23 DNA is at least 5 %, 10% or 20%, more preferred at least 30% or may even be as high as 50%, 75% or 100% (100 % being a complete demethylation in the region wherein the methylation status has been determined; i.e. all methylation sites in the healthy (steatosis) DNA have been demethylated) .
  • the diagnosis is, of course, the more accurate, the more methylation sites are included in the determination of the methylation status. It is therefore preferred to include at least 5, preferably at least 10, more preferred at least 20, es ⁇ pecially at least 30 methylation sites (i.e. sites which are methylated in KRT23 DNA of healthy persons) in the determination according to the present invention.
  • these methyla ⁇ tion sites are consecutive methylation sites in the KRT23 gene. It is also possible to examine more than one region with consec ⁇ utive methylation sites, e.g. in the promoter region and in one or more of the exons .
  • the methylation level for KRT23 according to the present invention is therefore reduced in a disease sample compared to a healthy, normal (steatosis) sample if preferably at least 5 %, 10% or 20%, more preferred at least 30% or even 50%, 75% or 100% of the methylation sites are demethylated in the disease (stea- tohepatitis) sample.
  • KRT23 methylation level is de ⁇ creased in a given detection method can preferably be estab ⁇ lished by analysis of a multitude of steatohepatitis samples with the given detection method. This can then form a suitable level from which the "decreased" status can be determined; e.g. by the above % difference or -fold change.
  • a decreased methyla ⁇ tion of the KRT23 gene is indicative for steatohepatitis.
  • a diagnostic assay for altered KRT23 methylation can be based on the detection of free circulating DNA the KRT23 promoter methylation in surgically resected or expiated human livers (comparison of normal liver with simple steatosis and steatohepatitis) . Based on the type of methylation patterns detected an assay for free circulating DNA can be established (e.g. as generally disclosed in WO 2008/103761 A2) . This assay can be for example a methylation- specific PCR or sequencing of specifically enriched free circu ⁇ lating DNA that has been treated with bisulfite.
  • a diagnostic assay in serum using KRT23 methylation as a signature for steatohepatitis is provided according to the present invention.
  • kits for determination of methylation of the KRT23 DNA in a sample for use in diagnosing steatohepatitis and/or for distinguishing between steatosis and steatohepatitis.
  • the present invention al ⁇ so relates to the use of a kit for determining the methylation level of KRT23 DNA in a sample, comprising determining the amount of demethylation of the KRT23 gene compared to a healthy methylation pattern for diagnosing a tissue sample or a sample of a body fluid for steatohepatitis .
  • the kit according to the present invention contains suitable methylation determination agents for KRT23 DNA methylation.
  • Such agents are well available for a person skilled in the art and depend on the very method applied for determination of the methylation status (e.g. MSP, use of restriction enzymes with activity governed by methylation status, use of antibodies specific for methylated nucleotide ba ⁇ ses, polynucleotide sequencing, bisulfite treatment and sequenc ⁇ ing, pyrosequencing, and AQAMA; see above) .
  • the diagnostic kit for di ⁇ agnosing steatohepatitis in a tissue sample or a sample of a body fluid according to the present invention comprises:
  • KRT23 methylation determination primer pair i.e. a KRT23 binding primer pair which defines a methylation region in the KRT23 gene, preferably a primer pair which is free of CpG mo ⁇ tifs, especially a primer pair which is free of single nucleo ⁇ tide polymorphisms (SNPs) and,
  • an agent containing bisulfite optionally (if the method includes a bisulfite step) , an agent containing bisulfite.
  • the kit according to the present invention in ⁇ cludes a KRT23 methylation determination primer pair which specifically binds to the KRT23 DNA and DNA amplifying reagents, such as a suitable PCR DNA polymerase and reagents (nucleotides, buffers, etc.) for performing PCR in connection with methylation determination.
  • DNA amplifying reagents such as a suitable PCR DNA polymerase and reagents (nucleotides, buffers, etc.) for performing PCR in connection with methylation determination.
  • These primers do not contain CpG motifs and flank the CpG islands of which the methylation status is to be deter ⁇ mined.
  • the primers thus bind in the genomic region of the K23 gene (Seq. ID. No. 1) or in the promoter region of the KRT23 gene (Seq. ID. No. 2) .
  • the primers are designed so as not to contain SNP loci (currently, the following 386 SNPs are known for the KRT23 gene (all sequences are included in Seq. ID. o.1; reference is therefore made to the positions in Seq. ID. No .1) :
  • Preferred primer pairs according to the present invention are designed to amplify a region in the KRT23 DNA which is at least 20 nucleotides, preferably at least 50 nucleotides, espe ⁇ cially at least 100 nucleotides long (nucleotide count without the nucleotides of the primer themselves) .
  • the size can also be significantly longer; maximum lengths are defined only by the technical practicality of the PCR method (if the ampli ⁇ fied sequence is too long for appropriate amplification and/or sequencing) .
  • the primer pairs are designed to amplify at least 5, preferably at least 10, more preferred at least 20, especially at least 30, methyla ⁇ tion sites of the KRT23 DNA (again the term "methylation sites refers to sites which are methylated in a healthy DNA) .
  • Methyla ⁇ tion occurs at cytosine bases on the DNA, usually where the cy- tosine is followed by a guanosine base (therefore being a "CpG- motif”) .
  • Possible methylation sites are therefore all sites hav ⁇ ing the nucleotide sequence "CG".
  • a sample of known steatohepatitis status e.g. a sample from a healthy individual
  • Such standard samples or other KRT23 standards are preferably further constituents of the present kit.
  • a gel electrophoresis or sequencing device or system detects the sequence which results from PCR amplifica ⁇ tion.
  • a kit for detecting the methylation level of KRT23 DNA in a sample comprising emulsion PCR beads (Williams et al., Nat. Methods 3 (2006), 545-550.
  • Emulsion PCR isolates indi- vidual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. A PCR then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing.
  • Emulsion PCR has been commercialized by 454 Life Sciences, is also known as "Polony sequencing" and "SOLiD sequencing” (developed by Agencourt, later Applied Biosystems, now Life Technologies) and allows amplification of complex gene libraries .
  • the kit can comprise suitable primer pairs specific for KRT23 DNA and/or reagents enabling methylation determination of KRT23 DNA in a given sample.
  • the kit may also contain instruc ⁇ tions for determining steatohepatitis diagnosis and prognosis based on the detection of the particular methylation degree or pattern according to the present invention.
  • the present kits are specifically useful for diagnosing a tissue sample or a sample of a body fluid for steatohepatitis.
  • kit may further include additional components such as additional oligonucleotides or primers and/or one or more of the following: buffers, reagents to be used in the assay (e.g., wash reagents, polymerases or internal control nucleic acid or cells or else) and reagents capable of detecting the presence of bound nucleic acid probe or primers.
  • buffers e.g., wash reagents, polymerases or internal control nucleic acid or cells or else
  • reagents capable of detecting the presence of bound nucleic acid probe or primers e.g., wash reagents, polymerases or internal control nucleic acid or cells or else
  • reagents capable of detecting the presence of bound nucleic acid probe or primers e.g., wash reagents, polymerases or internal control nucleic acid or cells or else
  • reagents capable of detecting the presence of bound nucleic acid probe or primers e.g., wash rea
  • kits and reagents of the above and foregoing are also covered by the present in ⁇ vention.
  • the kit may also include instructions regarding each particular possible diagnosis, prognosis, theranosis or use, by correlating a combined KRT23 DNA methylation level with a particular diagnosis, prognosis, theranosis or use, as well as in ⁇ formation on the experimental protocol to be used.
  • Figure 1 shows DNA amount that can be extracted from 1 ml patient plasma.
  • Figure 2 shows the KRT23 gene including methylation sites in the first exon in region chrl7: 39,092,678-39,093,032. Primers have been designed which include these methylation sites but do not contain CpG motifs or SNPs .
  • Figure 3 shows the purification of the PCR products.
  • PCR products were separated on agarose gels and purified
  • Bioan- alyzer Profile shows significant accumulation of the correct PCR product .
  • Figure 4 shows the correlation of KRT23 expression and KRT23 locus demethylation in liver. KRT23 expression and demethylation correlate and are highest in steatohepatitis .
  • Figure 5 shows the correlation of KRT23 demethylation with the pathological phenotype and KRT23 expression in liver. Evalu ⁇ ation of specific CpG sites allows an exact correlation of the pathological phenotype with demethylation of the KRT23 locus (KO: Control; S: Steatosis; SH: Steatohepatitis).
  • KRT23 is a marker suitable for differentiating between steatosis and steatohepatitis.
  • Expression of KRT23 is ac ⁇ tivated in steatohepatitis only; in liver tissue which only shows steatotic changes, no expression can be detected.
  • Measure ⁇ ment of KRT23 expression is based on the amount of m-RNA or pro ⁇ tein in liver tissue or body fluids.
  • differential diag ⁇ nosis can be achieved by determination of the methylation status of the KRT23 gene in samples containing DNA, for example blood derived samples containing free circulating DNA.
  • Gene expression is often regulated by methylation of DNA upstream of the start of genes or in the first exons of genes (Felsenfeld et.al. Nature 1982, PMID 7070505).
  • This DNA methyla ⁇ tion can be measured with several methods, for example by meth ⁇ ylation specific quantitative PCR (Zhang et.al., Int. J. Cancer, 2008, PMID 18546260), by sequencing bisulfite treated DNA (From- mer et.al., PNAS 1992, PMID 1542678) or by extraction of methylated DNA by antibody pulldown followed by high-throughput se ⁇ quencing (Weber et.al, Nat. Genet 2005, PMID 16007088).
  • Patient blood contains small amounts of free DNA that is shed into the bloodstream by tumors or other tissues. This free, circulating DNA can be investigated for relevant biomarkers, ke the changes in methylation pattern described here.
  • Free DNA was extracted from patient serum with the QIAamp Circulating Nucleic Acid Kit, Qiagen (Qiagen, Hilden, Germany) . All serum samples used in the project were collected from pa ⁇ tients after full consent and under a valid license from the lo ⁇ cal ethical committee. 5ml whole blood were collected in EDTA supplemented tubes. After centrifugation the plasma fraction was separated and stored at -80°C. 1ml aliquots were thawed and used for analysis. Usually approximately 20 to 40ng of free, circu ⁇ lating DNA could be extracted from 1ml of plasma (Fig. 1)
  • the region 6kb upstream of KRT23 and its full coding se ⁇ quence was screened for sites of methylation in the UCSC genome browser.
  • the first exon of KRT23 contains multiple methylation sites in the chrl7 : 39, 092, 678-39, 093, 032 region.
  • primers which amplify this region and the included CpG islands were designed using perlprimer software (PerlPrimer vl.1.21, Copyright ® 2003-2011 Owen Marshall) .
  • the primers had the following sequence:
  • KRT23-fwd GTGGTGAAGGATAGGGAGAT ( Seq . Id . No .3 )
  • KRT23-rev CCAAAAAATAAAACAAAACTCAAC ( Seq . Id . No .4 )
  • the target sequence could be amplified from bisulfite treat ⁇ ed DNA with this primer pair. Purification of PCR products
  • PCR products were bound to beads and amplified in an emulsion PCR using the Ion Torrent OneTouch system. After PCR reaction the template bearing beads were enriched and loaded on ⁇ to Semiconductor Chips which were then introduced into the Ion Torrent PGM and the sequencing reaction was started.
  • Next generation sequencing can produce very high numbers of reads in a single sequencing run. This allows to generate a ro ⁇ bust statistical value of the methylation status of a single CpG in the target sequence. In the present analyses between 2000 and 6000 reads were analyzed per sample.
  • sequence reads obtained for each sample were individual ⁇ ly compared to the reference sequence by a combination of perl scripts and ClustalW (Larkin M. et al. Bioinformatics 2007, PMID: 17846036) . After alignment the sequence of each CpG site in the target read was determined. The number of C and T reads was summarized over all reads and the percentage of thymidines at this position determined. This percentage represents the de- methylation of this CpG island in the free DNA from the patient serum.
  • KRT23 mRNA The expression level of KRT23 mRNA is different in steatosis and steatohepatitis , confirming our earlier results (Starmann et. al., PLoS One 2012, PMID 23071592).
  • DNA was extracted from liver tissue, bisulfite treated and the KRT23 locus was sequenced.
  • the demethylation of the KRT23 locus correlates very well with the expression level of KRT23 in the same tissues ( Figure 4) . This allows drawing the same pathological classification from the methylation patter of DNA as from the gene expression values of KRT23.
  • KRT23 expression levels from mRNA levels may necessitate a liver biopsy which is costly and implies possible complications. It is necessary to create a non-invasive assay to establish KRT23 as a biomarker of liver disease.
  • the measurement of DNA demethylation of the KRT23 gene and promoter from free, circulating plasma DNA presents such an assay.
  • KRT23 demethylation levels of select individual CpG sites demonstrate a high level of correlation to KRT23 expression in the liver and thus pathological classification.
  • Krt23 gene (chrl7: 39,078,952 - 39,099,836); preferred methyla ⁇ tion regions are given in bold; sequences used and amplified in the present examples are highlighted.

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Abstract

L'invention concerne l'utilisation de la méthylation du gène de kératine 23 (KRT23) en tant que marqueur pour permettre la distinction entre la stéatose et la stéatohépatite.
EP12813365.9A 2011-12-23 2012-12-21 Diagnostic de la stéatohépatite Withdrawn EP2794918A1 (fr)

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